这项研究是由西南德意志研究联合会（DFG WA 2804）和西南大学教育资助的实验和临床研究中心，最大的一个合作德尔布吕克分子医学中心在柏林的Charité医学院。资助者在研究设计，数据收集和分析，决定发表或准备手稿的作用。我们感谢罗伯特·韦斯特法尔先生在他的技术支持，在我们的实验室实习。

所有动物的程序，必须由当地机构执行前的动物福利委员会批准。于MR测量麻醉和生理监测（体温，呼吸频率）足够水平的必不可少的要求。 1。小鼠骨髓来源的树突状细胞生成<ol><li>提取C57BL / 6小鼠骨髓细胞如前所述，该协议的历史可以追溯到1992年13最初是由拉尔夫·斯坦曼（1943年至二○一一年），树突状细胞的发现者14组 12。 </li><li>简言之，提取后不久，胫骨和腓骨牺牲老鼠。下工作的层流罩（防止微生物污染），冲洗骨髓成一个小的陪替氏培养皿用洗涤培养基（5％FBS，1％青霉素/链霉素和1％HEPES的RPMI）12。 </li><li>通过细胞的细胞悬浮液转移斯特拉惯性（网目尺寸为100μm，尼龙）到无菌的50毫升离心管中，以去除骨和剩余的组织。经过多次洗涤步骤和裂解步骤12，活细胞使用台盼蓝和血球计数。 </li><li> 重悬在毫升-1（补充有10％FBS的RPMI 培养基中的浓度为400,000个细胞的骨髓细胞 ，1％青霉素/链霉素，1％HEPES和1％L-谷氨酰胺），含有75毫微克毫升-1鼠标粒巨噬细胞集落刺激因子（MGM-CSF）从293FT的小鼠GM-CSF的质粒转染的HEK细胞的上清液中获得。转移10ml的细胞悬浮液（4×10 6个细胞）在皮氏培养皿（100毫米×20毫米），在CO 2培养箱中培养（37℃，5％CO 2）。第3天，用10毫升新鲜培养基（MGM-CSF终浓度保持在75纳克毫升-1）补充旧的介质。第6天，取出10毫升的旧介质和补充用10毫升新鲜培养基（MGM-CSF终浓度= 75纳克毫升-1）。第8天，取出10毫升的旧介质，并补充10毫升新鲜培养基（MGM-CSF终浓度= 150毫微毫升-1）。 </li><li>第10天，1微克毫升-1脂多糖（LPS），24小时成熟分化的DC。 </li></ol> 2。标记树突状细胞与19架F-富<ol><li>在24小时熟化步骤（1.5），标签的DC与1毫米富氟全氟-15 - 冠醚颗粒（PFCE）（500-560纳米）也为24小时。准备大氟标记的粒子通过乳化PFCE，在Pluronic F-68（总体积2.5毫升）使用细胞破坏钛超声波发生器，采用连续脉冲程序（幅度100％，功率= 250 W），持续60秒。 </li><li>在上述培养步骤中，收获成熟和氟标记的DC使用组织培养细胞刮棒，转移到50毫升的离心管中，离心在500×g下在室温下5分钟。 </li><li>要洗去剩余的颗粒细胞和​​死细胞，留下收获细胞附着在聚-L-赖氨酸的菜肴。为了制备这一步，大衣的培养皿与聚-L-赖氨酸（1毫克毫升-1）在37℃下1小时，之后洗掉未结合的聚-L-赖氨酸用PBS。 </li><li>上述收集的细胞在无血清培养基中的聚-L-赖氨酸包被的板孵育，使细胞附着（1小时，在37℃下，5％CO 2）。 </li><li>洗掉培养基中，丢弃非贴壁细胞，其余的颗粒细胞和​​死细胞洗三次，用预温（37°C）的PBS。 </li><li>收获贴壁细胞治疗胰蛋白酶-EDTA（0.25％胰蛋白酶-EDTA，在37℃孵育3分钟）。 </li><li>另一个洗涤离心步骤后，重悬在无血清的无菌PBS将所需量的成熟的DC。 </li><li>为了判断细胞是否已成功标记，建议检查physicocheMICAL特性的细胞用流式细胞仪（FACS）。的侧向散射（SSC）揭示了一种增加的粒度在这些细胞中，如前面所述11。还应该记住的是，成纤维细胞可能污染的DC培养物，也可以采取19 F颗粒平行。因此，DC纯度应评估（使用流式细胞仪通过测量表面CD11c +细胞CD11b +细胞的百分比）前在体内应用。 </li></ol> 19 F-标记的树突状细胞在体内的应用3。 <ol><li>管理DC（10 6 - 10 7）在一个体积为50 - 100微升通过将鼠标定位在保持器中的C57BL / 6小鼠皮内注射到后肢并小心地插入到皮肤的上层的26½ģ针申请前（0.5毫升的结核菌素注射器针永久连接使用，以尽量减少死体积）。为了确保Ñ​​适当的皮内应用，重要的是，针的插入部分下方的皮肤是部分可见的。如果针是不可见的了，针已经插入过深进入皮肤层。对于未经训练的人，皮内的应用程序可以在麻醉下进行。 </li><li>图片四肢在体内的氟（第19楼）/质子（1H）磁共振成像（见下一节）注射后21小时。 </li></ol> 4， 在体内 19 F / 1ħ磁共振成像设立MR扫描的详 ​​细说明是指在Bruker MR扫描仪使用的的控制软件Paravison（版本5.1）。指的是供应商特定的功能和项目的名称已用斜体突出显示。对于其他MR扫描仪，这些步骤可能要进行调整，根据生产商的指引。 <ol><li>安排访问一个专门的小动物磁共振扫描仪。对于足够的图像质量，与磁场强度为9.4特斯拉以上且量身定制的射频线圈（ 例如 1 H / 19 F型双可调体积射频线圈，35毫米内径50毫米的长度;快速生物医学，维尔茨系统，德国）建议。 </li><li>此前MRI，准备麻醉和生理监测的核磁共振成像扫描仪的设置。确保的动物的呼吸用面罩床/保持器被连接到的异氟烷系统和温度探头和呼吸焊垫连接到远程动物监测系统（ 例如 SA仪器公司，型号1025，纽约，美国）。后者用来监视动物的重要的参数，例如体温和呼吸。 </li><li>麻醉小鼠用异氟醚的系统连接到鼠标室通过吸入麻醉状态。 0.2升分钟-1和0.1升分钟-1，分别调整空气流速和O 2和3％异氟醚（从蒸发器调整）约2分钟，直到所需的麻醉水平达到（以下脚趾捏没有响应）。最佳流速可能取决于正在使用的设置上。要知道，高流速可能导致脱水。上文所提到的，在任何时候都需要仔细监测动物的条件。 </li><li>将鼠标俯卧鼠标床/持有的小动物磁共振扫描仪（ 图1）。 </li><li>同时保持空气和O 2恒定的流速，调整异氟醚蒸发0.8 - 1.5％，直到达到最佳的呼吸模式。 50 - 70次每分钟的呼吸速率建议。 </li><li>在实验过程中保持身体的温度在36-37°C通过采用温水（或交替暖空气）循环系统。 </li><li>用无菌眼润滑软膏在测量过程中保持鼠标湿润的眼睛。 </li><li>在确保动物在成像保持器和连接到监测系统，移动夹持器的1 H / 19 F号RF线圈（即定位在动物扫描器磁体的等角点）的中心。 </li><li>鼠标等的设置位置，膝关节内的磁体的等角点，无论是一个自动化的方法（ 例如，使用一个马达驱动的动物保持器结合的定位激光），或通过手动计算需要移动的保持器的距离进磁体达到等角点。 </li><li>为了确认在扫描仪中的动物的正确定位和以后的规划形象片的几何形状（如大小，位置和旋转空间），收购侦察三种标准方向（轴向，冠状，矢状）使用快速和低分辨率图像的图像采集方法（ 如TriPilot协议，它采用了FLASH脉冲序列）。 </li><li>调谐射频线圈的1 H共振频率（ 例如400.1 MHz为9.4ŧ）和19 F的共振频率（ 例如 376.3 MHz为9.4ŧ）和匹配的线圈的特性阻抗50欧姆的动物MR扫描仪中使用的调谐监视器。调谐和匹配是必要的传输和信号接收达到最佳条件。 </li><li>执行必要的自动的系统设置，包括垫片来微调调谐射频鼠标（ 例如ADJ_SF）的拉莫尔频率和参考增益调整的RF振幅的均匀性的磁场（ 例如ADJ_SHIM），系统频率调整（ 如ADJ_REFG）。所有这些设置是尽可能地均匀，重要的是使磁性元件的静态和可变字段（B 0，B 1）的感兴趣的区域。 </li><li>沿三个正交方向采集的第二组的侦察员图像（如上述），之后，调整视场（FOV），使得两下肢芳E可见骨盆英尺（长×宽×高= 50x25x25毫米）。这些图像有助于规划的最终3D 1 H / 19 F扫描的方向。 </li><li>设置了TurboRARE的3D协议1 H-扫描:TR / TE = 1500/53毫秒，FOV = 50x25x25毫米，矩阵= 400x200x200，难得因数= 16（扫描时间约60分钟）。调整视场与侦察员图像的帮助下，开始扫描（ 交通灯 ）。 </li><li>与至少1,000毫秒的TR装入单脉冲FID序列。打开编辑扫描和设置核19楼"使用手动增益设置，取消选择"工具箱"的编辑方法自动参考收益。 </li><li>开始测量，而不记录数据实时显示MR信号通过GSP（去设置）。 19 F，如果频谱信号的采集窗口内太低，添加更多的平均值，在编辑的方法和/或调节的脉冲衰减器（TX0，否则SP0滑块），直到思此项目是清晰可见的。调整的基本频率，以中心19女性在0 Hz的谱峰在收购窗口。 应用基本频率 ，并按下停止 。请注意，在异氟醚麻醉作为可能会生成一个背景信号。在9.4特斯拉核磁共振成像之间的19 F-的含粒子和异氟醚的化学位移是1800赫兹左右。确保激发带宽小于3600赫兹，以避免激励异氟醚的19 F-标记的细胞或颗粒一起。上正确的19 F-标记的细胞所产生的信号的基本频率应设置。或者，另一种方法，可以用来作为麻醉实验者认为合适。 </li><li>克隆（副本）TurboRARE的3D协议，从以前的1 H扫描。转到编辑方法和取消选择自动的参考增益（如上）。将细胞核从编辑扫描 19女性 。改变矩阵128x64x64和稀土因数至少为40（最多64个）。对于TR / TE 1,000 / 6毫秒和64的平均值时，扫描时间是约70分钟。接收机增益设置为101（ 工具箱），并开始扫描，而不使用GOP（去管道）的任何进一步的调整。 </li><li>当扫描完成后收回的MR扫描仪鼠标座。小心拔下鼠标的持有人。如果鼠标不牺牲体外分析（ 如组织学或光谱，见下文）后直接MR测量，严密监控，直到它已完全从麻醉中恢复。体温调节是受麻醉，所以在恢复过程中，把鼠标被放置在一个温暖的温度调节垫在一个单独的笼子里。一旦鼠标已经完全从麻醉中恢复，它返回到其控股笼和动物室。 </li><li>要覆盖19 F 1 H图像的图像，使用" 查看"菜单 Ø佩蒂翁在同一软件（Paravison）的。 底图功能下可以找到关联 。保存底衬，加载的新形象，并通过定义一个新的颜色查找表突出的19个F层。要区分的19楼和1 H扫描所选择的颜色，改变阈值（ 切查表 ）19 F信号将有选择的颜色和背景1 H形象将留在灰度。其他后处理程序（ 如 ImageJ的）可用来创建的1 H / 19 F图像的覆盖。 </li><li>如果在体内 19 F信号的量化是必要的，遵循一个简单的定量协议如前所述15,16。简单地说，可以通过将管内的视场角下一个鼠标的PFCE乳液，使用一种已知浓度的参考量化。收购后的MR图像，全TIFY使用的感兴趣区域（ROI）的分析，19 F信号的强度，并比较在体外校准曲线， 如图2所示。 </li></ol> 5。淋巴结磁共振波谱（MRS） <ol><li>牺牲了鼠标后，取出腘窝淋巴结和小5毫米核磁共振管，并加入100微升2％的煤灰。 </li><li>安装内标的19 F光谱线圈（ 如环形线圈），它具有至少为5毫米的开口用于保持的NMR管中，淋巴结。 </li><li>线圈内的NMR管中放置和移动线圈的磁体的等角点。 </li><li>调谐射频线圈19 F共振频率（ 例如 376.3 MHz为9.4ŧ）和相匹配的线圈的特性阻抗50欧姆的动物MR扫描仪中使用的调谐监视器。调谐和匹配是必要的，以达到最佳的传输和信号条件接待处。 </li><li> 垫补工具箱 0内所有垫片参数设置和按申请。一般是没有必要用匀场的方法，由于样品的体积是相对小的。如果有足够的19 F信号是可用的，自动化的方法可施加垫片，系统频率和上面所描述的接收器的增益。 </li><li>与至少1,000毫秒的TR装入单脉冲FID序列。打开编辑扫描的细胞核，并设置19楼使用手动增益设置，取消选择"工具箱"的编辑方法自动参考收益。设置数的平均值为1。 </li><li>启动顺序使用GSP。 19 F，如果频谱信号的采集窗口内太低，在编辑的方法和/或添加更多的平均值调节的脉冲衰减器（TX0，否则SP0滑块），直到一个信号是清晰可见的。调整的基本频率，使19 F SPEctral峰是在0 Hz采集窗口的中心，并适用于基本频率。再次调整脉冲衰减器，使信号最大化，达到90°激发。当信号是最大的新闻停止 。 </li><li>设置在"编辑"，64和256之间的方法 （或更多的，如果必要，这取决于信号）的平均值和使用的GOP开始采集。检测到的信号线性相关与平均值的数目。在量化过程中牢记，检测到的信号需要进行归一平均。此外，已知浓度的校准标准或校准曲线是必要的信号（ 图2），归一化的19 F的细胞数来计算。 </li><li>一旦扫描完成后，取出样品，将样品和进行步骤5.3。 </li></ol>

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作者宣称，他们有没有竞争经济利益。

这种方法采用19 F / 1ħMRI跟随移动到淋巴结的DC提供了学习的机会， 体内的免疫细胞的迁移模式。树突状细胞是快速迁移的免疫细胞，是能够通过三维结构不紧密粘附的特异性底物17的情况下的机动的很好的例子。虽然所描述的技术的低空间分辨率（μm的范围内）是不与高的分辨率（nm范围内），使用这种技术，可以实现与多光子显微镜，它仍然是可能的，研究细胞迁移的性质， 在体内以及全球一段较长的时间。此外，显微镜，使其成为目前不适合大面积成像在活的有机体中有一个有限的穿透深度和有限的视场。 由于非侵入性的技术，有可能周一itor免疫细胞的迁移数天，在不牺牲鼠标经过调查。该技术的另一个优点是可以叠加的19 F的图像捕获与解剖1 H扫描的细胞迁移，从而使细胞在活体内精确定位。这种技术将使我们能够研究直流迁移的分子机制。与典型的在体外迁移实验中，这种方法使研究中的细胞迁移一个生理的3D环境。 19 F在MRS和MRI的潜在应用长期以来一直被认为7,18。高旋磁比，高自旋和天然同位素丰度19女性磁共振成像的理想人选7。另外，19 F和1 H（就其NMR属性）之间的相似性是有意义的一个先决条件吨之间的重叠他解剖1H MRI标记细胞MRI与19女性 。事实上1 H / 19 F 1 H / X核核磁共振成像MRI的一个优点是，相同的RF谐振器可用于19 F和1 H原子核。在大多数应用中，用于19 F / 1ħ核磁共振成像，体积谐振器时，可以调整到19 F和1 H。因此，由于B 1几乎相同的字段的两个通道，从1 H通道（即更容易测量）的19 F通道的传输功率设置是可能的。 与任何大小的颗粒（从超小的到大的微小尺寸的颗粒）标记细胞时，需要考虑的一个重要因素是生物操纵和毒性的潜力。我们最近表明，通过增加标签的颗粒的大小，我们可以促进DC的成熟状态11。一种可能的我们发现的解释是颗粒大于500 nm到被吞噬，而比19的内吞作用;前者吸收机制的优势定义DC的性质。其他的物理特性（ 例如，颗粒形状和表面的拓扑结构），也可以改变标记的细胞的生物功能，因此也应仔细考虑。对于任何给定的实验装置需要标签的DC与颗粒的，因此，它是强烈建议典型的细胞生物学检测并行地进行实验以确定/排除这些细胞的生物学特性的颗粒上的任何影响。可以进行的几个实验，根据实验研究的问题。要排除影响DC的成熟细胞表面标志物（ 如 CD80，CD86）表达的流式细胞仪，测量的建议。要排除影响抗原的摄取和演示，吞噬的experiments和T细胞的启动实验，分别建议。在迁移实验的情况下，表达的趋化因子（CC）的受体（如CCR7）和体外迁移检测的建议。 虽然19 F / 1ħMRI持有特别是用于临床研究的细胞迁移的承诺，目前有一些限制。这些包括:（i）的空间分辨率，排除直接可视化的单个细胞，（二）不足19女性灵敏度（检测限数10 5细胞内一个投资回报率），（三）增加扫描的结果持续时间平均增加补偿为以前的限制，（ⅳ）19 F RF市场上的谐振器（五）可能产生的不良背景19 F信号由其他含氟元素如isofluorane和聚四氟乙烯（PTFE）在MR ​​的硬件组件内（一个有限的范围如电容器和连接电缆）。 除了 ​​从磁场强度和梯度强度的因素，例如，一个主要的决定因素，决定空间分辨率的水平谐振器使用表21的无线频率（RF）的灵敏度。核磁共振成像的分辨率是密切相关的信号对噪声比（SNR）21，并减少体素大小扩增的空间分辨率时，在SNR的损失是可以预料的。而不会影响信号灵敏度提高空间分辨率的方法之一是采用低温冷却线圈，提高信噪比，减少热噪声22。使用的1H头线圈，其采用了低温系统的帮助下，我们可以在实验性自身免疫性脑脊髓炎的细胞浸润的最近的可视化，使用的是在图面的分辨率（35X35）μm2的23后。 19 F / 1小时MRI这个低温冷却技术的应用将是一个运算portunity克服一些与细胞信号检测和解决的MRI限制。 的MRI细胞在体内跟踪的一个重要的问题是一个活的有机体中的特定位置，这些细胞的定量。对于这一点，它是能够进行19 F MRS（见5.1-5.9）。通过采用所获得的19 F MRS不同数目的19 F-标记的直流测量的校准曲线，它是可以推导出到达的淋巴结的细胞的数目。 19 F MRS测量的淋巴结可以比作19 F MRS直流校准曲线。此外，通过比较从DC细胞与纯PFCE的校准曲线的MRS数据，我们可以推断出，这是可以检测到每个细胞约10 13 19 F自旋标记后。根据MR原则，最低检测限为10 18自旋每个体素24。因此，我们可以假设，我们是10 DC每个体素用9.4 T的磁共振成像能够检测到的最小数目。 总之，我们在这里描述的协议，它在体内的调查研究在病理生理过程中的细胞迁移是有利的。非侵入性的技术，使纵向研究，而不需要牺牲大量的动物;合成19 F的图像的能力，从1 H解剖图像上的标记的细胞的细胞迁移促进最佳的和高选择性的跟踪;和19 F MRS提供一个机会，判断定位在体内特定的解剖区域的细胞数。

细胞在体内的跟踪是在一些生物医药领域的一个重要方面。对于这一点，非侵入性的成像技术，可以选择性地本地化细胞过一段时间是极​​其宝贵的。三维磁共振成像（MRI）的发展之前，不限于显微分析或组织切片的免疫细胞迁移的跟踪。细胞跟踪的帮助下，MRI在过去的几年中取得了巨大的关注，不仅为免疫学研究的免疫细胞在体内的行为，同时也为临床和干细胞研究。在90年代中期，最初的研究氧化铁纳米粒子发起级联发展与MRI追踪细胞。氧化铁颗粒缩短MR弛豫时间（T2 *）标记的细胞，从而导致信号损耗MR图像。氧化铁颗粒已雇用标签的巨噬细胞，少突胶质细胞progenitors 3和许多其它类型的细胞。这些颗粒也被临床被FDA批准用于标记细胞疫苗在黑色素瘤患者4。由于在体内或体外与氧化铁颗粒标记的细胞依赖于缩短的T2 *信号，后者还可以带来体内的易感性相关T2 *的影响，如微出血，铁沉淀物或气泡，因此可能很难识别标记的细胞在体内的来自其他背景T2 *信号灭绝5。 在这篇文章中，我们描述了一种技术用于跟踪在体内的树突状细胞（DC）采用19 F / 1ħ磁共振成像（MRI）。这种细胞追踪技术才被引入几年后，于2005年6 19 F在MRI首次确认申请报告已7。其中一个重要的ADVA氧化铁颗粒细胞标记的19 F超过ntage是低的生物组织中发生的19 F，这使得它可以非常有选择性与基本上无背景图像跟踪细胞。此外，有可能获得从传统的1H核磁共振成像的解剖图像，叠加的19 F标记的细胞从移植的MR信号。因此，19 F / 1ħ核磁共振成像是相当有关的研究在体内细胞迁移。用这种方法研究的细胞都标有19架F-富。综合来自主要由碳原子和氟原子组成的全氟碳（PFC），通常用于制备颗粒。这些化合物是不溶于水和乳化之前， 在体外或体内应用。通常的PFC颗粒大小已受聘于其他群体在体内 19 F-MRI跟踪实验介于100纳米和245纳米6,8-10。然而，我们已经表明，标签与全氟-15 -冠-5 -醚（PFCE）随着粒径（&gt; 560nm的颗粒增加的树突状细胞中的效率）11

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>C57BL/6 mice</td><td>Charles River, Berlin</td><td></td><td></td></tr><tr><td>RPMI</td><td>Gibco</td><td>21875-091</td><td></td></tr><tr><td>FBS Superior</td><td>Biochrom AG</td><td>S 0615</td><td></td></tr><tr><td>HEPES</td><td>Gibco-Invitrogen</td><td>15630-056</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030-024</td><td></td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Sigma Aldrich</td><td>D8662</td><td></td></tr><tr><td>PFA</td><td>Santa Cruz</td><td>sc-281692</td><td></td></tr><tr><td>Perfluoro-15-crown-5-ether </td><td>ChemPur</td><td>391-1996</td><td></td></tr><tr><td>Pluronic F-68 </td><td>Sigma Aldrich</td><td>P5556</td><td></td></tr><tr><td>Petri dishes (35 x 10 mm)</td><td>VWR, Germany</td><td>391-1996</td><td></td></tr><tr><td>27 &amp;frac12; G syringes</td><td>VWR, Germany</td><td>612-0151</td><td></td></tr><tr><td>Nylon cell strainers (100 &amp;mu;m mesh) </td><td>VWR, Germany</td><td>734-0004</td><td></td></tr><tr><td>NMR tubes </td><td>VWR, Germany</td><td>634-0461</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>Dissection tools</td><td>FST</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator </td><td>Binder</td><td></td><td></td></tr><tr><td>Small animal MR system</td><td>Bruker Biospin</td><td></td><td>9.4T BioSpec 94/20 USR, ParaVision Acquisition and Processing Software</td></tr><tr><td>&lt;sup&gt;1&lt;/sup&gt;H/&lt;sup&gt;19&lt;/sup&gt;F dual-tunable volume RF coil </td><td>Rapid Biomed, W&amp;#252;rzburg, Germany</td><td></td><td>35 mm inner diameter, 50 mm length</td></tr><tr><td>&lt;sup&gt;19&lt;/sup&gt;F spectroscopy coil </td><td>in-house</td><td></td><td>tune/match loop coil, 4 turns, inner diameter 5 mm, 10 mm long, two capacitors for tuning and matching</td></tr><tr><td>Isoflurane inhalation system</td><td>F&amp;#246;hr Medical Instruments GmbH</td><td></td><td></td></tr><tr><td>Animal monitoring system Model 1025</td><td>SA Instruments Inc., New York, USA</td><td></td><td></td></tr></tbody></table>

monitoring dendritic cell migration using 19f / 1h magnetic resonance imaging

如磁共振成像（MRI）的非侵入性的成像方式的不断进步，极大地提高了我们的学习能力，在生物体的生理或病理过程。 MRI也证明是一个有价值的工具捕捉移植细胞在体内 。初始小区标记策略用于MRI使用的造影剂的影响的MR弛豫时间（T1，T2，T2 *）和引起增强（T1）或耗尽（T2 *）标记的细胞存在的信号。 T2 *增强剂，如超小型氧化铁剂（USPIO）已经被用来研究细胞迁移和一些人也已被FDA批准用于临床应用。 T2 *剂的缺点是难以区分从其他文物，如血块，微出血或气泡标记细胞的信号灭绝创建的。在这篇文章中，我们描述了一个新兴的技术跟踪细胞在体内基于氟（第19楼）丰富的颗粒细胞标记。这些颗粒的制备是通过乳化的全氟化碳（PFC）的化合物，然后用的标签细胞，其后，可以通过19 F核磁共振成像成像。的PFCs的重要的优点包括（i） 在体内的碳结合的19 F 在体内 ，然后通过19 F磁共振波谱产生背景图像和量化的细胞信号的完整的细胞选择性强（ⅱ）的可能性的情况下进行细胞跟踪。

18〜24小时后，皮内的应用程序，19 F-标记的树突状细胞（DC）迁移到排水的腘淋巴结。可以理解的DC，通过到排水politeal的淋巴结的淋巴管的运动，通过叠加的1 H 19 F DC图像（ 图2A）的解剖图像。我们以前曾报道这些细胞在体内的迁移，以及19 F-粒度对DC免疫生物学，包括吸收效率11的影响。为了量化的DC迁移到淋巴结的程度，我们提取引流淋巴结，并进行19 F MRS（ 图2B）。当我们比较19 F 19 F得到的信号从不同的DC标记的数目与的相同的PFCE粒子（校准曲线， 图2C），我们可以推断得到的信号从每个淋巴结19 F-标记的DC到达引流淋巴结的数目。在图2A和2B中所示的代表性的实验中，我们可以推断，3.6×10 5个负载抗原的DC达到7.5×10 4个DC抗原，未加载正确的淋巴结，而到达了正确的淋巴结。 <img alt="图1" src="/files/ftp_upload/50251/50251fig1.jpg" /> 图1。鼠标鼠标上的小动物磁共振扫描仪持有者的位置。 <img alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50251/50251fig2highres.jpg" src="/files/ftp_upload/50251/50251fig2.jpg" /> 图2。 （A）直流标有1毫米560纳米的粒子PFCE 19架F-标记的DC 在体内的迁移使用19 F MRS量化。<html> ES，加载（右）无抗原（左）和后肢皮内给药。全鸡卵清蛋白（OVA）作为抗原; OVA与DC一起温育19 F颗粒。树突状细胞显示为19 F MR信号（红色），而淋巴结和淋巴管中所示的1 H解剖MR图像（灰度）。图像被收购用19 F / 1 H两可调谐音量鸟笼谐振器（B）从小鼠的淋巴结在A的NMR管中被提取出来并放置所示。 19 F MRS（见协议文本）和振幅计算进行快速傅立叶变换（FFT）的收购自由感应衰减（FID）测定19 F信号（C）过了一段24小时，不同的大量的DC被打成1毫米560纳米的PFCE颗粒和19 F信号测定19 F刘健在BEF =的"https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50251/50251fig2large.jpg"目标="_blank"&gt;点击这里查看大图。

Watch this Scientific Journal Video about Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging at JoVE.com

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

细胞利用磁共振成像跟踪，在过去几年里取得了显着的关注。本协议描述标签的树突状细胞与氟（<sup&gt; 19</sup&gt; F）的丰富的颗粒，这些细胞在体内的应用程序，和监测他们迁移到引流淋巴结的程度<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H MRI和<sup&gt; 19</sup&gt; F刘健。

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

monitoring-dendritic-cell-migration-using-19f-1h-magnetic-resonance

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

11.8K 次观看.  A joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine. 细胞利用磁共振成像跟踪，在过去几年里取得了显着的关注。本协议描述标签的树突状细胞与氟（ 19 F）的丰富的颗粒，这些细胞在体内的应用程序，和监测他们迁移到引流淋巴结的程度 19 F / 1 H MRI和 19 F刘健。

视频: Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

注册生物成像，纳米材料可用于诊断和治疗监测

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

科研

生物工程

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

免疫与感染

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

医学

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

基于CFSE-试验鼠皮肤树突状细胞的迁移习进引流淋巴结在与感染<em&gt;牛分枝杆菌</em&gt;卡介苗

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 &#181;m/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

Here we describe methods using time-lapse, phase-contrast microscopy to image mouse resident peritoneal macrophages in a chemotactic complement C5a gradient. The protocols can be extended to other immune cells.

小鼠巨噬体化学术的延时成像

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a ...

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

使用正电子发射断层扫描对实验性自身免疫性脑脊髓炎小鼠模型中的 CD19+ B 细胞进行成像

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

监测树突状细胞迁移

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此视频中的章节

注册生物成像，纳米材料可用于诊断和治疗监测

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

在体内 ^19女性 MRI对细胞跟踪

跟踪小鼠骨髓单核细胞

成像的CD4 T细胞间质迁移发炎真皮

基于CFSE-试验鼠皮肤树突状细胞的迁移习进引流淋巴结在与感染

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

小鼠巨噬体化学术的延时成像

使用正电子发射断层扫描对实验性自身免疫性脑脊髓炎小鼠模型中的 CD19⁺ B 细胞进行成像